The use of synthetic peptide immunogens to produce antibodies of defined specificities is an important recent advance in immunochemistry. The present invention is directed to a group of synthetic peptide immunogens that can be used as reagents to elicit antisera against vertebrate or plant calmodulins.
Calmodulin and other members of the family of calcium-modulated proteins are major intracellular receptors for the element calcium, a substance of major importance in the regulation of many diverse cell processes such as motility, proliferation, and nutrient utilization. In addition, calcium is generally thought to be critical in the homeostasis of individual cells as well as whole organisms.
In order to elucidate the mechanisms of calcium control and the mechanisms of action of many hormones, regulatory agents, toxins, and pharmacological agents, it is necessary to examine cellular receptors for calcium, such as the protein calmodulin, and the means by which these receptors mediate the biological effects of calcium.
Calmodulin, a relatively small protein, exhibits multiple in vitro activities, although the physiological significance of a number of these activities remains obscure. However, calmodulin or calmodulin activity has been detected in most eukaryotic cells examined, and the amino acid sequence of the protein, as well as its in vitro activities, are highly conserved throughout vertebrate and invertebrate species, suggesting that calmodulin may play a fundamental role in mediating intracellular calcium-dependent effects.
Immunochemical techniques presently offer great potential for elucidating the exact role of calmodulin and other calcium-modulated proteins in cell function. For example, antisera elicited against calmodulin have become especially useful as immunodiagnostic reagents to differentially detect or purify calmodulin in supramolecular complexes. In addition, antisera of well-defined specificity are useful as molecular probes to define functional domains in calmodulin.
Unfortunately, our immunochemical studies on calmodulin were initially hindered by the inability to develop an anti-calmodulin serum of useful titer by merely injecting calmodulin into laboratory rabbits, a problem most likely resulting from the ubiquitous distribution of calmodulin throughout vertebrate and invertebrate species and the fact that its amino acid sequence differs minimally from species to species.
We overcame this problem in 1980 when, after testing a number of antigen modifications on calmodulin, we determined that the injection of performic acid-oxidized calmodulin into rabbits would result in antisera of useful titer reactive with either the oxidized or unoxidized molecule. Using one of the rabbit antisera, we proceeded to test peptide segments encompasssing the entire calmodulin molecule to locate one or more immunoreactive sites in the protein. See Van Eldik, L. J. and Watterson D. M. "Reproducible Production of Antiserum against Vertebrate Calmodulin and Determination of the Immunoreactive Site" Journal of Biological Chemistry Volume 256 (9), pp. 4205-4210 (1981). Upon completion of these studies we proposed than an immunoreactive site of calmodulin is contained in an 18 residue region (residues 127-144) within the COOH-terminal domain of calmodulin.
Further immunochemical studies of calmodulin involved our efforts to elucidate a specific and critical immunoreactive site within the proposed 18 residue region of the protein, the results of which were recently described in Van Eldik, et al. "Elucidation of a Minimal Immunoreactive Site of Vertebrate Calmodulin", Archives of Biochemistry and Biophysics Volume 227 (No. 2) pp. 522-533, (Dec. 1983). In this publication we reported that the heptapeptide segment asparagine-tyrosine-glutamic acid-glutamic acid-phenylalanine-valine-glutamine of vertebrate calmodulin (corresponding to amino acid residues 137 to 143) is as immunoreactive as the entire 148-residue protein with one antiserum. We also determined this heptapeptide segment to be the smallest calmodulin segment and the only 7-residue segment in region 135-145 of the protein capable of quantitive immunoreactivity with this anti-calmodulin serum. The analogous minimal immunoreactive site of plant calmodulin would be the heptapeptide segment asparagine-tyrosine-glutamic acid-glutamic acid-phenylalanine-valine-lysine, corresponding to residues 137-143 of the intact protein.
Recent advances in Immunochemistry involving the utilization of synthetic peptides to produce antibodies of defined specifities suggested the possibility that a synthetic peptide modeled after the immunoreactive-site heptapeptide of vertebrate or plant calmodulin would be effective as an immunogenic reagent to elicit antisera reactive with intact calmodulin. From a practical standpoint the ability to synthesize a peptide immunogen reagent as effective in eliciting anti-calmodulin serum as the protein itself, would greatly facilitate research in this area. Actual calmodulin is difficult to obtain, costly and in short supply.
In considering the possible design of a suitable peptide immunogen, we were guided in large part by the widely held assumption that a synthetic peptide containing an amino acid sequence corresponding to the immunoreactive region of a given protein will elicit antisera against the intact protein provided the peptide immunogen contains more than 6 amino acid residues, and provided the antigenic segment contained in the synthetic peptide would be surface-exposed if present in the actual protein from which it is derived. These prerequisites for the design of synthetic peptide immunogens were recently postulated by Lerner, R. A. (1982) Nature (London) Volume 299, pp. 592 to 596.
However, although the minimal immunoreactive heptapeptide site of calmodulin met both of the above requirements, we were unable to produce anti-calmodulin sera when we injected laboratory rabbits with a synthetic heptapeptide corresponding to the immunoreactive site of the protein. The synthetic heptapeptide was immunogenic in that it elicited an antibody response in the rabbits; however, the antibodies reacted with the synthetic heptapeptide but not with intact calmodulin. Van Eldik, et al. "Elucidation of a Minimal Immunoreactive Site of Vertebrate Calmodulin" (cited above). These findings suggested that the production of antisera against an intact protein using surface-exposed peptide regions is more complex than had been previously assumed.
Given our inability to use a synthetic version of the minimal immunoreactive site of calmodulin as an immunogen to elicit antisera against the whole molecule, we proposed that an effective design of synthetic peptide immunogens may require, in addition to minimum peptide length and surface exposure of the peptide segment in the intact protein, a presentation of the antigen region in the synthetic immunogen in an appropriate environment that most closely approximates the environment of the peptide segment within the intact protein.